Erythropoietin (EPO) is a heavily glycosylated acidic glycoprotein with a molecular weight of approximately 35,000. The protein consists of 166 amino acids and has a leader signal sequence of 27 amino acids which is removed in vivo during secretion from the host cell. The sequence encoding the unprocessed EPO is 579 nucleotides in length (Jacobs et al, 1985, Lin et al, 1985, U.S. Pat. No. 4,703,008 and WO 86/03520).
Erythropoietin is the principal hormone involved in the regulation and maintenance of physiological levels of erythrocytes in mammalian circulation and functions to promote erythroid development, to initiate hemoglobin synthesis and to stimulate proliferation of immature erythroid precursors. The hormone is produced primarily by the adult kidney and foetal liver and is maintained in the circulation at concentrations of about 10-20 milliunits/ml of serum under normal physiological conditions. Elevated levels of EPO, induced by tissue hypoxia, trigger proliferation and differentiation of a population of receptive progenitor stem cells in the bone marrow, stimulating hemoglobin synthesis in maturing erythroid cells and accelerating the release of erythrocytes from the marrow into the circulation.
Recombinant EPO has been used to successfully treat patients, including patients having anemia as a result of chronic renal failure. As EPO is the primary regulator of red blood cell formation, it has applications in both the diagnosis and treatment of disorders of red blood cell production and has potential applications for treating a range of conditions.
The urine of severely anaemic patients was, at one time, almost the sole source for the commercial isolation of EPO. U.S. Pat. No. 3,033,753 describes a method for obtaining a crude EPO preparation from sheep plasma. The preparation of monoclonal antibodies specific for human EPO provided a means for identifying EPO produced from EPO mRNA, for screening libraries and for cloning the EPO gene. Human EPO cDNA has been cloned and expressed in E. coli (Lee-Huang, 1984, Proc. Natl. Acad. Sci. 81:2708). Isolation of the human EPO gene using mixtures of short or long synthetic nucleotides as probes led to the expression of biologically active EPO in mammalian cells (Lin, 1985, Proc. Natl. Acad. Sci. 82:7580; Lin, WO 85/02610; Jacobs, et al., 1985, Nature (Lond.) 313:806; Goto et al., 1988, Biotechnology 6:67). Jacobs, et al., 1985, supra, described the use of plasmids containing EPO DNA which were not integrated into the chromosomes of the COS host cells, but replicated autonomously in the cells to many thousands of copies, thereby killing the cells. Thus the expression of EPO was only a transient phenomenon in these cells.
Lin, in U.S. Pat. No. 4,703,008, reported expression of the human EPO gene in COS-1 and CHO cells. However, attempts to use transfected cells as production vehicles for EPO have been hampered by the low levels of EPO expressed by transfected cells. Given the important applications of recombinant EPO, there is much interest in developing more efficient methods for the expression of EPO.
Lin in U.S. Pat. No. 4,703,008 reported methods to increase the low amounts of EPO produced by transfected CHO cells (e.g. 2.99 u/ml/3 days) by a process of gene amplification. Levels of approximately 1500 units EPO/10.sup.6 cells/48 hours were reported by Lin, following amplification. Gene amplification involves culturing cells in appropriate media conditions to select cells resistant to a selective agent, such as the drug methotrexate. Selection for cells resistant to methotrexate produces cells containing greater numbers of DHFR genes and passenger genes, such as the EPO gene carried on the expression vector along with the DHFR gene or transfected with the DHFR gene.
However, gene amplification is a very time consuming and labour intensive process. A major disadvantage of amplification is the inherent instability of amplified genes (McDonald, 1990, Crit. Rev. Biotech. 10:155). As it is usually necessary to maintain the amplified cells in the presence of toxic analogs to maintain high copy number, amplification may be inappropriate for large scale production due to the costs and toxicity of the selective agent. The high copy number of the DHFR-target transgene may also sequestor transcription factor, leading to a retardation of cell growth.
Genomic clones of human EPO have been used in attempts to develop stably transfected mammalian cell lines that secrete high levels of active erythropoietin (Powell, et al., 1986, Proc. Natl. Acad. Sci. 83:6465; Masatsuga, et al., European Patent Application Publication No. 0 236 059). In PCT Application WO 88/00241 Powell, describes the preparation of mammalian cell lines (COS-7 and BHK) transfected with the Apa I restriction fragment of the human EPO gene and selected for high expression by amplification.
Human EPO cDNA has also been expressed in mammalian cells (Yanagi, et al., 1989, DNA 8:419). Berstein, in PCT application WO 86/03520 describes the expression of EPO cDNA in various host cells, resulting in the secretion of up to 160 ng/ml of EPO into the medium after amplification. European Patent Application publication No. 0 267 678 discloses expression of recombinant EPO and secretion into the culture medium at levels of 600 units/ml.
A few scaffold attachment region (SAR) elements have been shown to increase the expression of reporter genes in transfected cells. SAR elements are thought to be DNA sequences which mediate attachment of chromatin loops to the nuclear matrix or scaffold. SAR elements are also known as MAR (matrix-associated regions) (reviewed by Phi-Van and Stratling, Prog. Mol. Subcell. Biol. 11:1-11, 1990). These elements will hereinafter be referred to as "SAR elements". SAR elements are usually 300 or more base pairs long, and they require a redundancy of sequence information and contain multiple sites of protein-DNA interaction. SAR elements are found in non-coding regions: in flanking regions or introns.
Stief, et al., (Nature 341:343-345, 1989) stably transfected chicken macrophage cells by constructs which contained the CAT gene either fused to the lysozyme promoter, or to the lysozyme promoter and the lysozyme enhancer. When the transcription units contained in both constructs were flanked on both sides by lysozyme 5' SAR elements (A elements), gene expression was increased about 10 times relative to transfectants, which contained the constructs lacking the SAR elements.
Phi-Van, et al., (Mol. Cell. Biol., 10:2302-2307, 1990) determined the influence of the SAR element located 5' to the chicken lysozyme gene (A element) on the CAT gene expression from a heterologous promoter (herpes simplex virus thymidine kinase promoter) in stably transfected heterologous cells (rat fibroblasts). The median CAT activity per copy number in transfectants was 10 times higher for the transcriptional unit flanked on both sides by A elements than for the transcriptional unit lacking SAR elements.
Klehr, et al., (Biochemistry, 30:1264-1270, 1991) stably transfected mouse L cells by different constructs containing the human interferon .beta. gene. When the construct was flanked by SAR elements, the gene transcription level was enhanced 20-30 fold with respect to the SAR-free construct, containing only the immediate regulatory elements.
However, the above-noted experiments have been limited to a very few examples of SAR elements, expressing mostly reporter genes, such as chloramphenicol acetyl transferase (CAT) or luciferase. SAR elements have not shown consistent results in their effect on the expression of target genes and some target gene sequences have been found to inhibit the effect of SAR elements (Klehr, et al., 1991, Biochemistry 30:1264).